The primary objectives of this project are the characterization of conformation and energetics of nonnative states of proteins, the exploration of pH and salt induced unfolding and the development of models that permit maximum structural information to be extracted from NMR experiments on unfolded states of proteins. The primary computational tools utilized for these studies will be continuum solvation methods and molecular dynamics. These objectives will be approached by addressing three major specific aims. 1. Energetic models for the unfolding profiles of analysis of unfolded states will be developed and tested through studies on single domain polypeptide fragments from Protein A and Protein G. Utilizing experimental NMR data on these systems together with existing simulation results for the folding free energy profiles, continuum models for protein folding/unfolding will be developed and assessed. Of particular interest will be the use of continuum models to provide insight into the energetics of unfolded states and their associated statistical weights. 2. Models for acid unfolded states apomyoglobin will be developed in collaboration with Dyson and Wright, using NMR. Utilizing continuum methods to assess conformation dependent pH changes and molecular dynamics approaches to expose structure and fluctuations under low pH conditions, the investigators will characterize the nature of the low pH nonnative states of apomyoglobin. They will also examine the backbone and sidechain coupling constants in the nonnative states by extensive comparison with simpler dipeptide models and NMR data from Dyson and Wright. 3. The influence of salt on the nonnative states of the all beta protein, plastocyanin, will be examined using continuum solvation methods and explicit molecular dynamics. The investigation will explore the changes in thermodynamic stability of apoplastacyanin as a result of changes in ionic strength using continuum models. From explicit MD studies, they will also explore changes in structure and dynamics as a result of these changes in salt concentration. The detailed models to be developed will be compared directly to the NMR results on these systems and will help the interpretation planning of collaborative efforts ongoing with the Dyson and Wright laboratories.